We studied synthesis, secretion and degradation of lipoprotein lipase (LPL) in cultured murine 3T3-L1 adipocytes using antiserum to bovine milk LPL raised in chickens. The antiserum proved effective for radioimmunoassay, immunoprecipitation and immunoinactivation of mouse LPL. The major radioactive protein precipitated by bovine-LPL antiserum from cells incubated with S-35 methionine had the same mobility on SDS-PAGE as bovine LPL (Mr = 47,000). Studies with S-35 methionine showed that newly synthesized LPL is catalytically active, and up to 50% is transferred immediately to cell surfaces in the presence of insulin. Active LPL accounted for less than 20% of LPL-like protein in cells. Rapid turnover of LPL was indicated by a half-life of 30 min for radiolabeled LPL in chase experiments and LPL-activity in cycloheximide-treated cells. Calculations showed that more than half of the enzyme was degraded in cells. A single recessive mutation, cld, on mouse chromosome 17 causes a deficiency of LPL activity. Mice homozygous for this defect develop lethal hyperchylomicronemia within 2 days after birth with plasma triacylglycerol conc as high as 20,000 mg/dl. Radioimmunoassays, using bovine-LPL antiserum, showed that the amounts of LPL-like protein in diaphragm muscle, heart and brown adipose tissue of cld/cld mice were 2-5 times larger than that in unaffected mice. In vivo incorporation of S-35 methionine into LPL-like protein in 40 min was normal or above normal in diaphragm muscle, heart, brown adipose tissue and liver of cld/cld mice. The major radioactive protein precipitated by bovine antiserum from all tissues in both groups of mice had the same mobility on SDS-Page as bovine LPL. Our findings show that tissues in cld/cld mice can synthesize normal amounts of LPL-like protein, but the protein has little catalytic activity. Turnover of LPL-like protein may also be impaired in tissues of cld/cld mice.